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Airway hyperresponsiveness (AHR) and inflammation are both observed in human and equine asthma. The aim of this study was to assess the timeline and relationship of both features at the subclinical onset of severe equine asthma (SEA). First, the repeatability of the pulmonary function test (PFT) using impulse oscillometry system, and the methacholine bronchoprovocation test (BPT) were assessed at a 1-day interval on six SEA horses in clinical remission and six control horses. Then, clinical and ancillary tests were performed before and after a 1-week low-dust environmental challenge, including weighted clinical score, respiratory endoscopy, bronchoalveolar fluid cytology, PFT, and BPT. Both PFT and BPT showed acceptable repeatability. No test allowed SEA horses in clinical remission to be distinguished from control, unlike in human patients. Because of the low-dust environment, no significant difference was observed in the results of clinical and conventional ancillary examinations after the challenge. However, SEA horses showed increased AHR after the environmental challenge. At that stage, no signs of inflammation or changes in pro-inflammatory cytokines profiles (quantification and gene expression) were observed, suggesting AHR is present at an earlier stage of equine asthma than airway inflammation. This feature indicates SEA could present in a different disease pathway than neutrophilic human asthma.

Asthma encompasses a spectrum of heterogenous immune-mediated respiratory disorders sharing a similar clinical pattern characterized by cough, wheeze and exercise intolerance. In horses, equine asthma can be subdivided into severe or moderate asthma according to clinical symptoms and the extent of airway neutrophilic inflammation. While severe asthmatic horses are characterized by an elevated neutrophilic inflammation of the lower airways, cough, dyspnea at rest and high mucus secretion, horses with moderate asthma show a milder neutrophilic inflammation, exhibit intolerance to exercise but no labored breathing at rest. Yet, the physiopathology of different phenotypes of equine asthma remains poorly understood and there is a need to elucidate the underlying mechanisms tailoring those phenotypes in order to improve clinical management and elaborate novel therapeutic strategies. In this study, we sought to quantify the presence of neutrophil extracellular traps (NETs) in bronchoalveolar lavage fluids (BALF) of moderate or severe asthmatic horses and healthy controls, and assessed whether NETs correlated with disease severity. To this end, we evaluated the amounts of NETs by measuring cell-free DNA and MPO-DNA complexes in BALF supernatants or by quantifying NETs release by BALF cells by confocal microscopy. We were able to unequivocally identify elevated NETs levels in BALF of severe asthmatic horses as compared to healthy controls or moderate asthmatic horses. Moreover, we provided evidence that BALF NETs release was a specific feature seen in severe equine asthma, as opposed to moderate asthma, and correlated with disease severity. Finally, we showed that NETs could act as a predictive factor for severe equine asthma. Our study thus uniquely identifies NETs in BALF of severe asthmatic horses using three distinct methods and supports the idea that moderate and severe equine asthma do not rely on strictly similar pathophysiological mechanisms. Our data also suggest that NETs represent a relevant biomarker, a putative driver and a potential therapeutic target in severe asthma disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Janssen, Tosi, Hego, Maréchal, Marichal and Radermecker.)

Alaskan sled dogs develop a particular metabolic strategy during multiday submaximal exercise, allowing them to switch from intra-muscular to extra-muscular energy substrates thus postponing fatigue. Specifically, a progressively increasing stimulus for hepatic glycogenolysis and gluconeogenesis provides glucose for both fueling exercise and replenishing the depleted muscle glycogen. Moreover, recent studies have shown that with continuation of exercise sled dogs increase their insulin-sensitivity and their capacity to transport and oxidize glucose and carbohydrates rather than oxidizing fatty acids. Carnitine and acylcarnitines (AC) play an essential role as metabolic regulators in both fat and glucose metabolism; they serve as biomarkers in different species in both physiologic and pathologic conditions. We assessed the effect of multiday exercise in conditioned sled dogs on plasma short (SC), medium (MC) and long (LC) chain AC by tandem mass spectrometry (MS/MS). Our results show chain-specific modification of AC profiles during the exercise challenge: LCACs maintained a steady increase throughout exercise, some SCACs increased during the last phase of exercise and acetylcarnitine (C2) initially increased before decreasing during the later phase of exercise. We speculated that SCACs kinetics could reflect an increased protein catabolism and C2 pattern could reflect its hepatic uptake for energy-generating purposes to sustain gluconeogenesis. LCACs may be exported by muscle to avoid their accumulation to preserve glucose oxidation and insulin-sensitivity or they could be distributed by liver as energy substrates. These findings, although representing a "snapshot" of blood as a crossing point between different organs, shed further light on sled dogs metabolism that is liver-centric and more carbohydrate-dependent than fat-dependent and during prolonged submaximal exercise., Competing Interests: The authors have declared that no competing interests exist.

Biologic therapies have transformed the management of psoriasis, but clinical outcome is variable leaving an unmet clinical need for predictive biomarkers of response. Here we perform in-depth immunomonitoring of blood immune cells of 67 patients with psoriasis, before and during therapy with the anti-TNF drug adalimumab, to identify immune mediators of clinical response and evaluate their predictive value. Enhanced NF-κBp65 phosphorylation, induced by TNF and LPS in type-2 dendritic cells (DC) before therapy, significantly correlates with lack of clinical response after 12 weeks of treatment. The heightened NF-κB activation is linked to increased DC maturation in vitro and frequency of IL-17 + T cells in the blood of non-responders before therapy. Moreover, lesional skin of non-responders contains higher numbers of dermal DC expressing the maturation marker CD83 and producing IL-23, and increased numbers of IL-17 + T cells. Finally, we identify and clinically validate LPS-induced NF-κBp65 phosphorylation before therapy as a predictive biomarker of non-response to adalimumab, with 100% sensitivity and 90.1% specificity in an independent cohort. Our study uncovers important molecular and cellular mediators underpinning adalimumab mechanisms of action in psoriasis and we propose a blood biomarker for predicting clinical outcome., (© 2021. The Author(s).)

The AHR is an environmental sensor and transcription factor activated by a variety of man-made and natural ligands, which has recently emerged as a critical regulator of homeostasis at barrier organs such as the skin. Activation of the AHR pathway downmodulates skin inflammatory responses in animal models and psoriasis clinical samples. In this study, we identify CYP1A1 enzymatic activity as a critical regulator of beneficial AHR signaling in the context of skin inflammation. Mice constitutively expressing Cyp1a1 displayed increased CYP1A1 enzymatic activity in the skin, which resulted in exacerbated immune cell activation and skin pathology, mirroring that observed in Ahr-deficient mice. Inhibition of CYP1A1 enzymatic activity ameliorated the skin immunopathology by restoring beneficial AHR signaling. Importantly, patients with psoriasis displayed reduced activation of the AHR pathway and increased CYP1A1 enzymatic activity compared with healthy donors, suggesting that dysregulation of the AHR/CYP1A1 axis may play a role in inflammatory skin disease. Thus, modulation of CYP1A1 activity may represent a promising alternative strategy to harness the anti-inflammatory effect exerted by activation of the AHR pathway in the skin., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

FK228 (romidepsin) and suberoylanilide hydroxamic acid (vorinostat) are histone deacetylase inhibitors (HDACi) approved by the US Food and Drug Administration for cutaneous T-cell lymphoma (CTCL), including the leukemic subtype Sézary syndrome. This study investigates RAD23B and STAT3 gene perturbations in a large cohort of primary Sézary cells and the effect of FK228 treatment on tyrosine phosphorylation of STAT3 (pYSTAT3) and RAD23B expression. We report RAD23B copy number variation in 10% (12/119, P ≤ 0.01) of SS patients, associated with reduced mRNA expression (P = 0.04). RAD23B knockdown in a CTCL cell line led to a reduction in FK228-induced apoptosis. Histone deacetylase inhibitor treatment significantly reduced pYSTAT3 in primary Sézary cells and was partially mediated by RAD23B. A distinct pattern of RAD23B-pYSTAT3 co-expression in primary Sézary cells was detected. Critically, Sézary cells harboring the common STAT3 Y640F variant were less sensitive to FK228-induced apoptosis and exogenous expression of STAT3 Y640F, and D661Y conferred partial resistance to STAT3 transcriptional inhibition by FK228 (P ≤ 0.0024). These findings suggest that RAD23B and STAT3 gene perturbations could reduce sensitivity to histone deacetylase inhibitors in SS patients., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

We report the first macrocycle-based ratiometric molecular thermometer exploiting the conformational thermosensitivity of a calixarene functionalized with two different fluorophores. Thanks to the dependence on temperature of the efficiency of excitation energy transfer between the organic fluorophores, the thermometer works over a 60 °C-wide temperature range with a sensitivity of 4% °C -1 .

Formalin is toxic and has recently been classified as carcinogenic leading to a proposed European formalin ban. But, the pathology use of formalin has however been completely overlooked, and this is proving to be a relevant issue, as no alternative, reliable, tissue fixative is available. Various systems have been proposed to reduce formalin use and exposure; long-term storage and disposal of formalin is also a problem. With this in mind, under vacuum sealing (UVS) systems have been proposed for transportation/storage, however, for how long tissue retains its characteristics (morphological and molecular) is unknown. This study aims to compare histology specimens stored by formalin immersion (FI) and specimens stored after fixation with UVS technique with no additional formalin, at different time periods. Twenty tissue samples (10FI; 10UVS) were stored for different time periods (15 days, 1-2-3-6-12 months) for a total of 120 samples, compared with regard to their morphology, histochemistry, immunoreactivity (24 specific antibodies) and DNA status. All samples showed well-preserved morphology and overlapping staining quality. A significant reduction in immunoreactivity was however identified in the various time periods, particularly for heat pre-treated nuclear antigens, and this commenced earlier (1 month) for FI. UVS storage showed higher DNA content than FI but slightly poorer DNA integrity. These results add important knowledge to the use of UVS in daily practice, as long-term storage of pre-fixed tissue in UVS is not detrimental to the quality of tissue while having the boon of using very little formalin with less operator exposure and lower disposal costs.

Polysaccharide storage myopathy (PSSM) is a widely described cause of exertional rhabdomyolysis in horses. Mitochondria play a central role in cellular energetics and are involved in human glycogen storage diseases but their role has been overlooked in equine PSSM. We hypothesized that the mitochondrial function is impaired in the myofibers of PSSM-affected horses. Nine horses with a history of recurrent exercise-associated rhabdomyolysis were tested for the glycogen synthase 1 gene (GYS1) mutation: 5 were tested positive (PSSM group) and 4 were tested negative (horses suffering from rhabdomyolysis of unknown origin, RUO group). Microbiopsies were collected from the gluteus medius (gm) and triceps brachii (tb) muscles of PSSM, RUO and healthy controls (HC) horses and used for histological analysis and for assessment of oxidative phosphorylation (OXPHOS) using high-resolution respirometry. The modification of mitochondrial respiration between HC, PSSM and RUO horses varied according to the muscle and to substrates feeding OXPHOS. In particular, compared to HC horses, the gm muscle of PSSM horses showed decreased OXPHOS- and electron transfer (ET)-capacities in presence of glutamate&malate&succinate. RUO horses showed a higher OXPHOS-capacity (with glutamate&malate) and ET-capacity (with glutamate&malate&succinate) in both muscles in comparison to the PSSM group. When expressed as ratios, our results highlighted a higher contribution of the NADH pathway (feeding electrons into Complex I) to maximal OXPHOS or ET-capacity in both rhabdomyolysis groups compared to the HC. Specific modifications in mitochondrial function might contribute to the pathogenesis of PSSM and of other types of exertional rhabdomyolyses.

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator. Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 10 7 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage. This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Background: Besnoitiosis is caused by different species of intracellular protozoan parasites belonging to the family Sarcocystidae and affecting multiple host species worldwide. Including B. besnoiti, ten species are described infecting animals. Among ungulates, Besnoitia bennetti infects horses, donkeys and zebras and was described in Africa and in the USA where donkey besnoitiosis is considered as an emerging disease., Case Presentation: A two-year-old male donkey was purchased in May 2016 in poor body condition (cachexia, alopetic areas and pruritus mainly on neck and head) by the present owner in Le Roeulx (Belgium) from a milk producing donkey farm in Frasnes-lez-Buissenal (Belgium). Shortly after its purchase and shearing, the donkey presented with crusts, hyperkeratosis (both flanks and neck) anorexia and cachexia. A treatment with phoxim was given with no improvement. A cutaneous biopsy of hyperkeratotic skin was performed in July. It showed a perivascular eosinophilic infiltrate with a large thick walled cyst located in the dermis containing numerous bradyzoites. This was highly suggestive of besnoitiosis. Several skin biopsy samples were obtained for qPCR analysis and confirmed the presence of Besnoitia spp. DNA. Further laboratory diagnosis tests were performed (western blot and rDNA sequencing) confirming Besnoitia bennetti aetiology for the male. For the female, the punch-biopsy, haematology and qPCR were negatives but the western blot showed the presence of antibodies directed to Besnoitia spp. Further clinical examination performed in August highlighted scleral pinhead sized cysts (pearl) in the right eye and between nares. Another ten-year-old female donkey purchased in France and sharing the same accommodation showed a good clinical condition, but a thorough clinical examination showed the presence of numerous cysts on the inner face of upper labial mucosa. A daily treatment based on sulfamethaxzole and trimethoprim (Emdotrim 60% Mix®, 30 mg/kg) was given orally and some improvement was noticed., Conclusion: This is the first evidence of Besnoitia bennetti infection (Protozoa: Apicomplexa) in donkeys (Equus asinus) in Belgium.

B cells participate in immune surveillance in human circulation and tissues, including tumors such as melanoma. By contrast, the role of humoral responses in cutaneous immunity is underappreciated. We report circulating skin-homing CD22+CLA+B cells in healthy volunteers and melanoma patients (n = 73) and CD22+ cells in melanoma and normal skin samples (n = 189). Normal and malignant skin featured mature IgG and CD22 mRNA, alongside mRNA for the transiently-expressed enzyme Activation-induced cytidine Deaminase (AID). Gene expression analyses of publically-available data (n = 234 GEO, n = 384 TCGA) confirmed heightened humoral responses (CD20, CD22, AID) in melanoma. Analyses of 51 melanoma-associated and 29 normal skin-derived IgG sequence repertoires revealed lower IgG1/IgGtotal representation compared with antibodies from circulating B cells. Consistent with AID, comparable somatic hypermutation frequencies and class-switching indicated affinity-matured antibodies in normal and malignant skin. A melanoma-associated antibody subset featured shorter complementarity-determining (CDR3) regions relative to those from circulating B cells. Clonal amplification in melanoma-associated antibodies and homology modeling indicated differential potential antigen recognition profiles between normal skin and melanoma sequences, suggesting distinct antibody repertoires. Evidence for IgG-expressing B cells, class switching and antibody maturation in normal and malignant skin and clonally-expanded antibodies in melanoma, support the involvement of mature B cells in cutaneous immunity.

Differentiation between Sézary syndrome and erythrodermic inflammatory dermatoses can be challenging, and a number of studies have attempted to identify characteristic immunophenotypic changes and molecular biomarkers in Sézary cells that could be useful as additional diagnostic criteria. In this European multicenter study, the sensitivity and specificity of these immunophenotypic and recently proposed but unconfirmed molecular biomarkers in Sézary syndrome were investigated. Peripheral blood CD4(+) T cells from 59 patients with Sézary syndrome and 19 patients with erythrodermic inflammatory dermatoses were analyzed for cell surface proteins by flow cytometry and for copy number alterations and differential gene expression using custom-made quantitative PCR plates. Experiments were performed in duplicate in two independent centers using standard operating procedures with almost identical results. Sézary cells showed MYC gain (40%) and MNT loss (66%); up-regulation of DNM3 (75%), TWIST1 (69%), EPHA4 (66%), and PLS3 (66%); and down-regulation of STAT4 (91%). Loss of CD26 (≥80% CD4(+) T cells) and/or CD7 (≥40% CD4(+) T cells) and combination of altered expression of STAT4, TWIST1, and DNM3 or PLS3 could distinguish, respectively, 83% and 98% of patients with Sézary syndrome from patients with erythrodermic inflammatory dermatoses with 100% specificity. These additional diagnostic panels will be useful adjuncts in the differential diagnosis of Sézary syndrome versus erythrodermic inflammatory dermatoses., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Sézary syndrome (SS) is a leukemic variant of cutaneous T-cell lymphoma (CTCL) and represents an ideal model for study of T-cell transformation. We describe whole-exome and single-nucleotide polymorphism array-based copy number analyses of CD4(+) tumor cells from untreated patients at diagnosis and targeted resequencing of 101 SS cases. A total of 824 somatic nonsynonymous gene variants were identified including indels, stop-gain/loss, splice variants, and recurrent gene variants indicative of considerable molecular heterogeneity. Driver genes identified using MutSigCV include POT1, which has not been previously reported in CTCL; and TP53 and DNMT3A, which were also identified consistent with previous reports. Mutations in PLCG1 were detected in 11% of tumors including novel variants not previously described in SS. This study is also the first to show BRCA2 defects in a significant proportion (14%) of SS tumors. Aberrations in PRKCQ were found to occur in 20% of tumors highlighting selection for activation of T-cell receptor/NF-κB signaling. A complex but consistent pattern of copy number variants (CNVs) was detected and many CNVs involved genes identified as putative drivers. Frequent defects involving the POT1 and ATM genes responsible for telomere maintenance were detected and may contribute to genomic instability in SS. Genomic aberrations identified were enriched for genes implicated in cell survival and fate, specifically PDGFR, ERK, JAK STAT, MAPK, and TCR/NF-κB signaling; epigenetic regulation (DNMT3A, ASLX3, TET1-3); and homologous recombination (RAD51C, BRCA2, POLD1). This study now provides the basis for a detailed functional analysis of malignant transformation of mature T cells and improved patient stratification and treatment., (© 2016 by The American Society of Hematology.)

In this work, the dynamics of electronic energy transfer (EET) in bichromophoric donor-acceptor systems, obtained by functionalizing a calix[4]arene scaffold with two dyes, was experimentally and theoretically characterized. The investigated compounds are highly versatile, due to the possibility of linking the dye molecules to the cone or partial cone structure of the calix[4]arene, which directs the two active units to the same or opposite side of the scaffold, respectively. The dynamics and efficiency of the EET process between the donor and acceptor units was investigated and discussed through a combined experimental and theoretical approach, involving ultrafast pump-probe spectroscopy and density functional theory based characterization of the energetic and spectroscopic properties of the system. Our results suggest that the external medium strongly determines the particular conformation adopted by the bichromophores, with a direct effect on the extent of excitonic coupling between the dyes and hence on the dynamics of the EET process itself., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Methylthioadenosine phosphorylase (MTAP) and the tumor suppressor genes CDKN2A-CDKN2B are frequently deleted in malignancies. The specific role of MTAP in cutaneous T-cell lymphoma subgroups, mycosis fungoides (MF) and Sézary syndrome (SS), is unknown. In 213 skin samples from patients with MF/SS, MTAP copy number loss (34%) was more frequent than CDKN2A (12%) in all cutaneous T-cell lymphoma stages using quantitative reverse transcription PCR. Importantly, in early stage MF, MTAP loss occurred independently of CDKN2A loss in 37% of samples. In peripheral blood mononuclear cells from patients with SS, codeletion with CDKN2A occurred in 18% of samples but loss of MTAP alone was uncommon. In CD4(+) cells from SS, reduced MTAP mRNA expression correlated with MTAP copy number loss (P < 0.01) but reduced MTAP expression was also detected in the absence of copy number loss. Deep sequencing of MTAP/CDKN2A-CDKN2B loci in 77 peripheral blood mononuclear cell DNA samples from patients with SS did not show any nonsynonymous mutations, but read-depth analysis suggested focal deletions consistent with MTAP and CDKN2A copy number loss detected with quantitative reverse transcription PCR. In a cutaneous T-cell lymphoma cell line, promoter hypermethylation was shown to downregulate MTAP expression and may represent a mechanism of MTAP inactivation. In conclusion, our findings suggest that there may be selection in early stages of MF for MTAP deletion within the cutaneous tumor microenvironment., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Emerging evidence suggests pathological and immunoregulatory functions for IgG4 antibodies and IgG4 + B cells in inflammatory diseases and malignancies. We previously reported that IgG4 antibodies restrict activation of immune effector cell functions and impair humoral responses in melanoma. Here, we investigate IgG4 as a predictor of risk for disease progression in a study of human sera (n = 271: 167 melanoma patients; 104 healthy volunteers) and peripheral blood B cells (n = 71: 47 melanoma patients; 24 healthy volunteers). IgG4 (IgG4/IgG total ) serum levels were elevated in melanoma. High relative IgG4 levels negatively correlated with progression-free survival (PFS) and overall survival. In early stage (I-II) disease, serum IgG4 was independently negatively prognostic for progression-free survival, as was elevation of IgG4 + circulating B cells (CD45 + CD22 + CD19 + CD3 - CD14 - ). In human tissues (n = 256; 108 cutaneous melanomas; 56 involved lymph nodes; 60 distant metastases; 32 normal skin samples) IgG4 + cell infiltrates were found in 42.6% of melanomas, 21.4% of involved lymph nodes and 30% of metastases, suggesting inflammatory conditions that favor IgG4 at the peripheral and local levels. Consistent with emerging evidence for an immunosuppressive role for IgG4, these findings indicate association of elevated IgG4 with disease progression and less favorable clinical outcomes. Characterizing immunoglobulin and other humoral immune profiles in melanoma might identify valuable prognostic tools for patient stratification and in the future lead to more effective treatments less prone to tumor-induced blockade mechanisms.

Despite recent discoveries of genetic variants associated with autoimmunity and infection, genetic control of the human immune system during homeostasis is poorly understood. We undertook a comprehensive immunophenotyping approach, analyzing 78,000 immune traits in 669 female twins. From the top 151 heritable traits (up to 96% heritable), we used replicated GWAS to obtain 297 SNP associations at 11 genetic loci, explaining up to 36% of the variation of 19 traits. We found multiple associations with canonical traits of all major immune cell subsets and uncovered insights into genetic control for regulatory T cells. This data set also revealed traits associated with loci known to confer autoimmune susceptibility, providing mechanistic hypotheses linking immune traits with the etiology of disease. Our data establish a bioresource that links genetic control elements associated with normal immune traits to common autoimmune and infectious diseases, providing a shortcut to identifying potential mechanisms of immune-related diseases., (Copyright © 2015 Elsevier Inc. All rights reserved.)